Antibodies are protein molecules having a structure based on a unit comprising four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulphide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, comprising one or more so-called C-domains. The N-terminal regions of the heavy and light chains, also known as V-domains, are variable in sequence and determine the specificity of the antibody. The regions in the variable domains of the light and heavy chains (V.sub.L and V.sub.H respectively) responsible for antigen binding activity are known as the hypervariable or complementarity determining regions (CDR). Natural antibodies have at least two identical antigen-binding sites defined by the association of the heavy and light chain variable regions.
It is known that proteolytic digestion of an antibody can lead to the production of antibody fragments. Such fragments, or portions, of the whole antibody can exhibit antigen binding activity. An example of a binding fragment is an F.sub.ab fragment which comprises a light chain associated with the V.sub.H and C.sub.H1 domains of a heavy chain. The bivalent F(ab.sup.1).sub.2 fragment comprises two such F.sub.ab fragments connected together via the hinge region, giving two antigen binding sites. F.sub.v fragments, consisting only of the V-domains of the heavy and light chains associated with each other may also be obtained. These F.sub.v fragments are monovalent for antigen binding. Smaller fragments such as individual V-domains (domain antibodies or dABs, Ward et al Nature, 341, 544 (1989) and individual CDR's (Williams et al, Proc. Natl. Acad. Sci, U.S.A., 86, 5537 (1989)) have also been shown to retain the binding characteristics of the parent antibody although generally most naturally occurring antibodies need both a V.sub.H and V.sub.L to retain full immunoreactivity.
Antibody fragments comprising V.sub.H and V.sub.L domains associated together to have antigen binding activity have also been described. The single chain F.sub.v fragment (scFv) comprises a V.sub.H domain linked to a V.sub.L domain by a flexible polypeptide linker such that the domains can associate to form an antigen binding site (see, for example, EP-B-0281604, Enzon Labs Inc).
Microbial expression systems for producing active antibody fragments are known in the literature. The production of Fab in various hosts such as E. coli. (Better et al, Science, 240, 104, (1988)), yeast (Horwitz et al, Proc. Natl. Acad. Sci, U.S.4, 85, 8678 (1988)) and the filamentous fungus Trichoderma reesei (Nyyssonen et al, Bio/Technology, 11, 591 (1993)) have previously been described, for example. It is also known that plants can be used as hosts for the production of SCFv fragments (Owen et al, Bio/Technology, 10, 790 (1992)) as well as whole antibodies.
An advantage of using antibody fragments rather than whole antibodies in diagnosis and therapy lies in their smaller size. They are likely to be less immunogenic than whole antibodies and more able to penetrate tissue. A disadvantage associated with the use of fragments such as the F.sub.ab, F.sub.v, and S.sub.c F.sub.v antibody fragments described above, however is that they have only one binding site for antigen binding as compared to the two or more sites contained in the whole antibody, preventing polyvalent binding to the antigen and hence leading to reduced avidity.
In an attempt to overcome this problem, attention has been directed to providing multivalent antigen binding proteins, that is binding proteins having more than one antigen binding site. In addition, there has been interest in producing antigen-binding proteins having multiple specificities capable of binding to different antigenic determinants and containing antigen binding domains derived from different sources. Antigen-binding proteins having distinct binding specificities may be useful, for example, in targeting effector cells to target cells by virtue of the specific binding of the different binding domains. By way of illustration, a bispecific antigen binding protein having specificity for both tumour cells and cytotoxic drugs may be used to target specifically cytotoxic drug to tumour cell in an efficient manner. By avoiding the need for chemical modification, adverse immune responses may be avoided.
Hitherto, the potential application of multivalent and multispecific antigen binding proteins have been hindered by the difficulties in generating and purifying such molecules.
Recombinant antigen-binding proteins having two binding sites may be prepared by methods such as chemical cross-linking of cysteine residues, either through cysteine residues introduced at the C-terminus of the V.sub.H of an F.sub.V (Cumber et al, J.Immunol., 149, 120 (1992)), through the hinge cysteine residues in F.sub.ab to generate (Fab.sup.1).sub.2 (Carter et al, Bio/Tech., 10, 163 (1992)) or at the C-terminus of the V.sub.L of an scFv (Pack and Pluckthun, Biochemistry, 31, 1579 (1992)). Alternatively, the production of bivalent and bispecific antibody fragments based on the inclusion of F.sub.ab fragments of C-terminal peptide sequences which promote dimerisation has been described. (Kostelny et al, (1992) J.Immunol., 148, 1547).
Bivalent or bispecific antibody fragments comprising a binding complex containing two polypeptide chains, one comprising two heavy chain variable domains (V.sub.H) in series and the other comprising two light chain variable domains (V.sub.L) in series are described in our pending European Patent Application No. 95307332.7.
Multivalent and/or multispecific antibody fragments are described in WO 94/09131 (Scotgen Limited). Specific binding proteins having two binding regions, contained at least in part on first and second polypeptide chains which chains additionally incorporate associating domains capable of binding to each other causing the polypeptide chains to combine are disclosed therein. It is disclosed that the first and second binding regions preferably are antibody antigen-binding domains, for example comprising V.sub.H and V.sub.L regions contained in a Fab fragment or in a single-chain Fv fragment, or may be derived from just one of the V.sub.H or V.sub.L regions of an antibody. The associating domains may suitably be derived from an antibody and may be inter alia antibody V.sub.H and V.sub.L regions. It is further disclosed that using a V.sub.H /V.sub.L domain combination to achieve association leads to the creation of a supplementary Fv domain such that the antibody produced may be trivalent. Schematic representations of the arrangements suggested in WO 94/09131 to produce trivalent fragments are shown in FIG. 1A. WO 93/11161 (Enzon Inc) describes multivalent antigen-binding proteins comprising two or more single-chain protein molecules, each single chain molecule comprising first and second polypeptides each comprising the binding portion of the variable region of an antibody heavy or light chain with the polypeptides being linked together via a peptide linker. Hypothetical trimers and tetramers are discussed, comprising three or four single-chain antigen binding proteins as appropriate. Schematic representations of the trivalent arrangements suggested are shown in FIG. 1B.
WO 91/19739 (Celltech Limited) discloses multivalent antigen binding proteins comprising an Fv fragment bound to at least one further Fv fragment by a connecting structure which links the Fv fragments together but which maintains them spaced apart such that they can bind to adjacent antigenic determinants. Conveniently the connecting structure consists of a spacing polypeptide and a linkage unit such as a cross-linking maleimide linker or a molecule which allows for non-covalent binding. Particularly preferred connecting structures which are disclosed are based on antibody joining and hinge region sequences.